Inactivation of the Open Reading Frame slr0399 in Synechocystis sp. PCC 6803 Functionally Complements Mutations near the QA Niche of Photosystem II

Abstract

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in QA function, has an apparently mobile QA, and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the originalpsbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion ofslr0399 restores photoautotrophy in D2R8 and in otherpsbDI mutants impaired in QA function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around QA in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes. The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in QA function, has an apparently mobile QA, and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the originalpsbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion ofslr0399 restores photoautotrophy in D2R8 and in otherpsbDI mutants impaired in QA function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around QA in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes. photosystem I photosystem II polymerase chain reaction the primary electron-accepting plastoquinone in PS II the second electron-accepting plastoquinone in PS II 3-(3,4-dichlorophenyl)-1,1-dimethylurea tetramethyl-p-benzoquinone (duroquinone) plastoquinone phylloquinone kilobase pair(s) high performance liquid chromatography isoflavone reductase-like protein N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid The cyanobacterium Synechocystis sp. PCC 6803 is a useful molecular genetic system to study oxygenic photosynthesis and cell physiology of photosynthetic microorganisms. The organism is unique in that it combines several desirable properties: (i)Synechocystis sp. PCC 6803 is spontaneously transformable and incorporates exogenous DNA into its genome via double-homologous recombination (1Grigorieva G. Shestakov S. FEMS Microbiol. Lett. 1982; 13: 367-370Crossref Scopus (218) Google Scholar, 2Williams J.G.K. Methods Enzymol. 1988; 167: 766-778Crossref Scopus (845) Google Scholar); (ii) the strain can grow (photo)heterotropically, thus enabling the creation of Synechocystis sp. PCC 6803 strains impaired in photosystem I (PS I)1 and/or photosystem II (PS II) function (for a recent review, see Ref. 3Vermaas W.F.J. Methods Enzymol. 1998; 297: 293-310Crossref PubMed Scopus (40) Google Scholar); and (iii) the entire genome sequence of Synechocystis sp. PCC 6803 is known (4Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 185-209Crossref PubMed Scopus (240) Google Scholar). Because of these properties, a variety of molecular genetic approaches have been applied to study the role of particular proteins in this organism. One of these approaches is the mapping and characterization of pseudorevertants, which carry second-site mutations restoring viability under conditions that are lethal for the original mutants. In the case of mutants that are impaired in photosynthesis, pseudorevertants with improved photosynthetic function and with secondary mutations in genes coding for proteins with known function have been isolated and analyzed (for example, see Refs. 5Nixon P.J. Rögner M. Diner B.A. Plant Cell. 1991; 3: 383-395Crossref PubMed Scopus (54) Google Scholar, 6Kless H. Vermaas W. J. Biol. Chem. 1995; 270: 16536-16541Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 7Chen Z.X., Yu, W.Z. Lee J.H. Diao R. Spreitzer R.J. Biochemistry. 1991; 30: 8846-8850Crossref PubMed Scopus (45) Google Scholar, 8Thow G. Zhu G. Spreitzer R.J. Biochemistry. 1994; 33: 5109-5114Crossref PubMed Scopus (32) Google Scholar, 9Yu J. McIntosh L. Methods Enzymol. 1998; 297: 18-27Crossref Scopus (5) Google Scholar, 10Girard-Bascou J. Pierre Y. Drapier D. Curr. Genet. 1992; 22: 47-52Crossref PubMed Scopus (55) Google Scholar, 11Wu H.Y. Kuchka M.R. Curr. Genet. 1995; 27: 263-269Crossref PubMed Scopus (21) Google Scholar). These genes may be identical to the ones carrying the original mutations or may be at a different locus. In eukaryotic genetic systems such as Chlamydomonas reinhardtii or Arabidopsis thaliana mapping of a site of a suppressor mutation in an unrelated gene is a time-consuming and complicated project. with the of a known in Synechocystis sp. PCC 6803 the of mapping of genes that second-site mutations has been and of a of with W.F.J. Methods Enzymol. 1998; 297: 293-310Crossref PubMed Scopus (40) Google Scholar). In this a gene can be the DNA the for In the study have applied this to photoautotrophic pseudorevertants of the D2R8 mutant S. Vermaas W.F.J. Biochemistry. 1998; PubMed Scopus Google which has primary mutations in the of the D2 protein that is of the PS II reaction the of these to to an open reading frame coding for a protein of an function. be in this suggest that Slr0399 may function as a to QA into its function has been for folding of proteins in Refs. PubMed Scopus Google and R.J. Curr. Biol. PubMed Scopus Google is known about the of in folding of protein and in insertion of Synechocystis sp. PCC 6803 were at in R. J. M. J. Microbiol. Google with at the of with with and The PS II to for of mutants with in PS II in to for photoautotrophic the of Synechocystis sp. PCC 6803 were and at for with of of of with of were and in and to a of at for to and were at for to cell DNA several with and with a to with DNA in and to a of The for and in a at to the to the DNA in and in and of the DNA with and a in and the in for and in with with and each of the into each a DNA each were the as were at at for in a at for and at for The for PCC 6803 D2R8 mutant were to about as a were to and of this a the the of each DNA different each at a different were applied the cell and to were of at in the at The of in a at a of were in with with or and The a and a and for PS II in a binding as in Ref. W.F.J. J. G. Scopus Google and of the were in a as in Ref. H. Vermaas W. J. Biol. 1995; PubMed Scopus Google One sp. PCC 6803 in growth and and were isolated as in Ref. Vermaas W. S. A. 1998; PubMed Scopus Google The quinone in isolated with in the for and other were with for at in the under with to Ref. A. M. G. PubMed Scopus Google The of in the to and the were at for to the The in the under and were in of a a with a of in the the of the to for a with at a of A. E. S. Lett. PubMed Scopus Google Scholar). The of the at a The were with the as a for of the of quinone were and analyzed in and with were the of with in of Scholar, of of the and quinone The Synechocystis sp. PCC 6803 mutant D2R8 not grow to mutations in the of the D2 protein and that the of QA and QA to be apparently mobile and other quinones S. Vermaas W.F.J. Biochemistry. 1998; PubMed Scopus Google Scholar). mutant second gene coding for the D2 and has mutations to a V247M/A249T/M329I mutation the D2R8 mutant in the of photoautotrophic were to The to which about or of than the of that for mutants. In to the genetic of the photoautotrophic of the D2R8 the gene photoautotrophic D2R8 and and in the mutations and in the photoautotrophic strains were Moreover, no mutations were in that isolated the these the pseudorevertants to the D2R8 mutant to that the site of in the strains located of DNA isolated the pseudorevertants the D2R8 mutant to photoautotrophy with high that the of D2R8 to photoautotrophic growth is to a genetic We of different to be in Synechocystis sp. PCC Several genes or gene coding for PS II proteins that have to the QA site and were DNA of the These were for their to the original mutant to of these genes to D2R8, that not the site of the secondary In to the or for the in the a mapping of with applied W.F.J. Methods Enzymol. 1998; 297: 293-310Crossref PubMed Scopus (40) Google of the sp. PCC 6803 that has been for the The DNA isolated and to of DNA with of the and a of the of the into DNA of and in a and DNA and to the original D2R8 mutant as under The to photoautotrophic growth as the DNA in each of the D2R8 that these the carrying the secondary mutation no with DNA with and may that a site of these is to the of the secondary not a to recombination in Synechocystis sp. PCC or (ii) the the secondary mutation these than or than and not in the of the secondary mutation in with sp. PCC 6803 of DNA of the the of the of the DNA of these D2R8 to photoautotrophic The and genome of the the secondary mutation were the of been for the other of the DNA of these D2R8 to photoautotrophic The and genome of the the secondary mutation were the of been for the other of the DNA of these D2R8 to photoautotrophic The and genome of the the secondary mutation were the of been for the other of the DNA of these D2R8 to photoautotrophic The and genome of the the secondary mutation were the of been for the other in a The of the that to of the original mutant were with the of the entire PCC 6803 W.F.J. Methods Enzymol. 1998; 297: 293-310Crossref PubMed Scopus (40) Google in to a in the genome that this unique to with the for each of the to DNA to a of the genome the site of the this this pseudorevertants and and the wild into the and and to the D2R8 Indeed, the pseudorevertants, but not the wild the original mutant to shown in the open reading and ones T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 185-209Crossref PubMed Scopus (240) Google and have no significant similarity to other open reading in the Slr0399 is similar to the to be encoded an open reading frame in of is similar to the protein encoded in In to which of these open reading the site of the the in and were an and in a In both the to be a ofslr0399 pseudorevertants and that and mutations this open reading to acid substitutions and a deletion the for and which also a to the of the 60% of Slr0399 in These suggest that inactivation of slr0399 in the D2R8 mutant leads to of PS II function in this to inactivation of slr0399 restore PS II function in PS II or this limited to the mutant D2R8 this the and were to different sp. PCC 6803 strains with mutations or The of this are in and that several different mutations in the of D2 and can be to photoautotrophic growth inactivation of slr0399 in Synechocystis sp. PCC the of in the mutant which has no PS II and no that photoautotrophic growth can be a secondary mutation in mutants with some PS II of different PS II mutants with and which the mutant slr0399 D2R8 pseudorevertants, and with wild type of in to photoautotrophic growth slr0399 mutants of the to has been and the of has been in Ref. in are the mutations in the D2R8 strain and W. and W. and W. and W. and W. and W. in mutant in Ref. mutants in Ref. in in in in The of the to has been and the of has been in Ref. S. Shestakov S. Vermaas W. Plant Biol. 1996; 30: PubMed Scopus Google These are the mutations in the D2R8 strain S. Vermaas W.F.J. Biochemistry. 1998; PubMed Scopus Google S. and W. in Ref. H. M. McIntosh L. M. Biochemistry. 1994; 33: PubMed Scopus Google in Ref. E. G. Vermaas W.F.J. Biochemistry. PubMed Scopus Google in a with DNA not in of the strains the PS II mutation other than the QA that in PS II Slr0399 the strains carrying mutations a the in D2R8 also be that Slr0399 may with the QA site as a than with the that deletion of a of Slr0399 leads to photoautotrophic growth in PS II mutants with at the QA a in which a near the of slr0399 the effects of this deletion and/or insertion the of located of DNA that but the at the site to the of slr0399. were to the Synechocystis sp. PCC 6803 wild the D2R8 and the D2 mutant for to The of the as Synechocystis sp. PCC 6803 genome of to is targeted inactivation of slr0399 photoautotrophic growth in the mutants D2R8 and slr0399 inactivation no the of the wild type The photoautotrophic growth the PS II the of and the of binding the of PS II for the a and the of in wild type slr0399 Moreover, the as as the of of PS II with not effects of the slr0399 deletion photoautotrophic growth PS II the of binding and the in the wild type and mutant strains of Synechocystis sp. PCC 6803 with or binding site binding site of wild in a PS II of the D2R8 and mutants were of the slr0399 inactivation of the wild The photoautotrophic of the deletion mutants than that of the wild and the of PS II in both D2 mutants to about of that in the wild of the slr0399 inactivation the of and in the mutants D2R8 and to to in the wild type of a ofslr0399 no phenotypic in D2R8, or the wild type not that the phenotypic effects in the D2R8 pseudorevertants and in the D2 mutant strains with slr0399 are in inactivation than to effects of genes that to be The D2R8 mutant has an apparently mobile QA in of PS II for of in this mutant is Moreover, in the of of in D2R8 is and the recombination have S. Vermaas W.F.J. Biochemistry. 1998; PubMed Scopus Google Scholar). this the QA of the strain were in in the strain the for the of about which is an of than that for the D2R8 is a in the strain than in wild quinones that in the D2R8 mutant S. Vermaas W.F.J. Biochemistry. 1998; PubMed Scopus Google in the strain not Moreover, the strain not and the of with D2R8. In the of the the of and recombination with the PS II the of in the were similar to in the wild type of in the and of in the D2R8 and mutants and the wild type strain with or a were the of a an the second and the of the to a of and are the were about that the of D2R8 is and in this strain the is to about in a a were the of a an the second and the of the to a of and are the were about that the of D2R8 is and in this strain the is to about wild type inactivation ofslr0399 not for D2R8. In the mutant and other PS II were as not as as in D2R8, and were to wild type inactivation and The are of an of QA function in D2 but not in wild inactivation of slr0399. One to to this is that the slr0399 gene the quinone and/or the of quinone in the of the organism. this may be the were isolated the D2R8, wild strains and of the wild type and strain is in no these the of phylloquinone been in the The of the other strains also similar not and phylloquinone were as sp. PCC similar to has been in other G. M. S. Scopus Google Scholar, S. K. Methods Enzymol. 1988; 167: Scopus Google Scholar). were deletion that the of the of PS II function in pseudorevertants is not to of a quinone different than at the QA The that Slr0399 is not involved with quinone but a role of this protein as a in plastoquinone insertion into photosystem II a Slr0399 of and is similar to the protein of function Ycf39 that is encoded in the cyanelle genome L. C. W. H. J. and D. A. and in the chloroplast genomes of A. R. A. C. and C. J. J. Biol. 1982; PubMed Scopus Google J. Plant Biol. 1995; 13: Scopus Google and A. and K. is also similar to the of an A. thaliana nuclear gene similarity and located H. M. E. A. A. J. T. L. H. W. K. and C. of Slr0399 and its is in of high similarity Slr0399 and Ycf39 proteins are the The in Slr0399 is a motif near the N terminus of the protein a which is in binding proteins (for review, see Ref. J. 1996; PubMed Scopus (218) Google and which with the of the or of Slr0399 the known of D. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). a sequence and at in The of a or at the of the motif and the a suggest that Slr0399 than NADH Biochemistry. Scopus Google Scholar, C. J. to Scholar). Moreover, a motif in G. H. G. J. Biol. PubMed Scopus Google is not in the that as a in this The of this with the motif to the of the protein sequence that are not in the cyanobacterium or in this protein is Slr0399 is to in the in Synechocystis sp. PCC 6803 Ycf39 is to be located in the chloroplast and to not be into the The of a chloroplast sequence in the A. thaliana Ycf39 protein Refs. G. J. J. PubMed Scopus Google Plant Biol. 1998; Scopus Google is in with into the chloroplast not Slr0399 and its Ycf39 are Slr0399 has a that is to the to for the protein secondary as of and J. J. Biol. 1982; PubMed Scopus Google M. E. I. G. A. PubMed Google and T. S. S. 1998; PubMed Scopus Google Scholar). but in Ycf39 this it is that this is an this is to an and this protein is to not the a of the in this is that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around QA in photosystem II. of chaperone-like proteins leads to a S. Genet. 27: PubMed Scopus Google Scholar). the in to the of the wild type Synechocystis sp. PCC 6803 strains were at at and were and to at the is to the for photoautotrophic growth of Synechocystis sp. PCC 6803 wild type C. L. Plant Scholar). The photoautotrophic growth of both strains to similar and as as at about cell and the strain at a The of growth of the strain to the of cell than to the of at the growth of the wild type and strains a of the cell in the a to cell not The of strain to photoautotrophic growth at not to inactivation of the PS II of not the of growth in the of about to cell at that Slr0399 to a function other than PS II as Indeed, are in both photosynthetic and respiratory and a role of Slr0399 that PS II is not of with W.F.J. Methods Enzymol. 1998; 297: 293-310Crossref PubMed Scopus (40) Google in this for the of a has with the of the entire Synechocystis sp. PCC 6803 genome sequence (4Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 185-209Crossref PubMed Scopus (240) Google its the of this transformable cyanobacterium to exogenous DNA into its genome recombination (1Grigorieva G. Shestakov S. FEMS Microbiol. Lett. 1982; 13: 367-370Crossref Scopus (218) Google J.G.K. Methods Enzymol. 1988; 167: 766-778Crossref Scopus (845) Google Scholar). of this in Synechocystis sp. PCC 6803 is a and mapping the of secondary mutations in pseudorevertants, as as of mutations and mutations via the of this is a for of is In the case of the this of photoautotrophic In D2R8 pseudorevertants that have been photoautotrophic growth to secondary mutations in an open reading frame the Synechocystis sp. PCC 6803 deletion of the entire ofslr0399 also photoautotrophic growth in D2R8. The D2R8 pseudorevertants and the PS II as with D2R8 and the of QA were to to wild type and inactivation of slr0399 also photoautotrophic growth and PS II in some other mutants that around the and that retained some other PS II mutants not be inactivation of slr0399 suggesting a Slr0399 and the QA site of PS II. The Synechocystis sp. PCC 6803 open reading is to a which high similarity with hypothetical protein Ycf39 is that the in chloroplast genomes in to the nuclear genome in of protein a motif and has an similarity with of One is the of isoflavone reductase-like proteins that are in different N. S. T. Yamada Y. Plant Cell. 1994; PubMed Scopus Google Scholar, E. S. E. M. D. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. C. R. S. Plant Cell. 1996; PubMed Scopus Google Scholar, A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Plant Biol. 33: PubMed Scopus Google Scholar, A. S. E. S. E. Plant Biol. 1998; PubMed Scopus Google Scholar). Slr0399 is identical and similar to proteins the of similarity to other of is not Slr0399 also similarity and with the that has been as a subunit of a the of its sequence similarity with the subunit of the complex I G. T. R. M. M. R. 1998; PubMed Scopus Google Scholar). The proteins have been in a the of their high sequence similarity with isoflavone which the of and which are involved in of in in to R. Y. G. Plant Biol. 1991; PubMed Scopus Google Scholar, K. D. M. W. J. 1991; PubMed Scopus Google Scholar, 1995; Scopus Google Scholar). proteins have been in that not in to A. in and in and are to have have been to function in to in Arabidopsis E. S. E. M. D. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google in S. A. C. R. S. Plant Cell. 1996; PubMed Scopus Google and to in A. S. E. S. E. Plant Biol. 1998; PubMed Scopus Google Scholar). has been that isoflavone reductase-like proteins are NAD(P)H as a which have that may or may not be to N. S. T. Yamada Y. Plant Cell. 1994; PubMed Scopus Google Scholar, E. S. E. M. D. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The similarity of Slr0399 with and with subunit of the complex I of and J. D. H. Lett. 1991; PubMed Scopus Google Scholar, M. J. 1991; PubMed Scopus (32) Google may be as these proteins may with The subunit of complex I in has no in the respiratory NADH The function of this subunit The that inactivation of slr0399 can different mutations in the binding of the D2 protein and not mutations in several other PS II to that Slr0399 may be involved with or into PS II QA or with of the or the of the The of Slr0399 in quinone or (for example, to of quinones that can to the QA site in the D2R8 mutant and that are at this is that no significant or in sp. PCC 6803 strains deletion not a role of Slr0399 in or in in the mutant the of the of phylloquinone not phylloquinone in in is to be with PS I in a of quinone PS I complex Y. K. S. Res. PubMed Scopus Google Scholar, W. Lett. Scopus Google Scholar). that the of PS phylloquinone is in the the of and is no for a decrease in the of PS I cell in the mutant not is that Slr0399 is involved in insertion of of the into PS I as but the of phenotypic of slr0399 deletion the that this phylloquinone is for the of PS II function in D2R8 and inactivation of slr0399 be that deletion of slr0399 the of the to of QA function in the mutants. of in the and of PS I not significant inactivation of slr0399 not suggesting that the of the system is not Moreover, is no that Slr0399 is a of PS II, it that Slr0399 with QA, or The that is that Slr0399 is a chaperone-like protein that is involved in not QA insertion into PS II the quinone to the reaction in a particular In wild this leads to reaction but in strains with mutations in the QA the D2 protein not QA in in such strains can be in in the of Slr0399 has been or this or no protein in QA assembly in this mutant system is the of this of Slr0399 as a protein in insertion of is the that the slr0399 inactivation mutant has a The that this in the of no PS II is for that the function of Slr0399 is not limited to assembly of PS II, but may other such as PS I and the complex I. In the Synechocystis sp. PCC 6803 are other open reading proteins that are similar to the of and similarity of the with and and The molecular of and are and proteins a near the N suggesting that these hypothetical proteins are or but may Slr0399 in their function. We are to and for and We for and of and for with of protein